Performance of malaria rapid diagnostic test in febrile under-five children at Oni Memorial Children’s Hospital in Ibadan, Nigeria, 2016

Introduction The World Health Organization (WHO) recommends testing of suspected malaria cases before treatment. Malaria rapid diagnostic test (mRDT) has been recommended for this purpose in endemic countries where microscopy is not accessible. However, its diagnostic performance remains a concern in clinical settings. We assessed diagnostic performance of RDT among febrile under-five children (U5) presenting at Oni Memorial Children's Hospital, Ibadan (OMCH). Methods A cross-sectional study was conducted among 370 febrile U5 attending OMCH February to May, 2016. We examined their finger prick blood samples for malaria parasitaemia using CareStartTM histidine rich protein II (HRP-2) RDT and microscopy. The sensitivity, specificity, positive and negative predictive values (PPV, NPV), false positive (FP), invalid rates (IR), likelihood ratio of positive and negative tests (LRP and LRN), were calculated. Results Mean age of the children was 28.17 ± 15.59 months. Malaria prevalence was 21.6% and 15.1% by mRDT and microscopy, respectively. Sensitivity of CareStartTM HRP-2 RDT was 94.6% (95% confidence interval (CI): 84.2-98.6), specificity: 91.4% (CI: 87.6-94.2), PPV: 66.3% (CI: 54.7-76.2), NPV: 98.9% (CI: 96.8-99.7), FPR 6.5%, IR 8.1%, LRP:10.6 and LRN:0.1. Conclusion Diagnostic performance of CareStartTM used in the study met the ≥ 95% sensitivity at 100 parasites/µL recommended by WHO. This finding provides clinical evidence that testing before anti-malarial treatment as recommended by WHO will identify cases of malaria infection and reduce unnecessary use of drugs. Healthcare workers should be educated on diagnostic accuracy of mRDT and adhere to the WHO's test-treat strategy for anti-malaria therapy.


Introduction
Malaria is a life threatening tropical disease responsible for significant morbidity and mortality especially, among children and pregnant women, with 48 to 304 million new cases of malaria infections reported annually worldwide. Nigeria accounted for up to 29% of the total estimated malaria cases and 26% of deaths in the African region [1]. Accurate diagnosis of malaria still remains a challenge because of lack of pathognomonic symptoms, as fever which is one of the commonest symptoms of the disease in children has numerous causes. Infection with bacteria, viruses, protozoa or fungi can manifest as febrile illnesses, thereby presenting with common overlapping manifestations making clinical diagnosis very difficult and leading to undue delay in initiating treatment of fever in children, with the attendant complications including deaths [2]. In the tropics and malaria-endemic regions, most fevers are presumed to be due to malaria and are treated empirically as such [3]. There is mounting evidence that a significant proportion of these febrile illnesses are non malaria [4,5]. These non-malarial febrile illnesses have been defined as infectious diseases in patients who present with undifferentiated fever and require malaria rapid diagnostic tests or microscopy, but in whom these tests were negative [6]. Despite the awareness of non malaria causes of febrile illness, empirical treatment of fevers, with antimalarial medicines continues in resource-poor settings. In order to limit the development of resistance to drugs and mitigate the consequences of failure of therapy, the World Health Organization advocate for test-based management of malaria and restricting artemisinin-based combination therapy (ACT) to only parasitologically confirmed cases [7]. Diagnosis of malaria based on blood film microscopy using thick and thin smears stained with Giemsa has remained the gold standard for many years [8,9]. However, the inherent limitations of microscopy such as non-availability of technical expertise and erratic electricity supply and dependence on sophisticated equipment have severely hindered its universal routine use particularly in a busy outpatient clinic and rural settings [10].
This necessitated the introduction of mRDT kits in the early 1990s with the potential to overcome the weaknesses associated with microscopy [11]. Malaria rapid diagnostic tests are based on malaria antigen detection and are meant to differentiate malaria from nonmalarial febrile illness, with a turnaround time of less than thirty minutes and have been shown to be effective [12].
CareStart TM malaria an HRP2-based immunochromatographic test which detects HRP2 antigen that is specific for Plasmodium falciparum has a very high performance level as reported by the most recent mRDT evaluation programme [13] and is among the mRDTs approved for procurement by National Malaria Elimination Programme (NMEP). Nigeria adopted mRDT as a diagnostic tool, where microscopic diagnosis is not feasible [14], however, the diagnostic performance of the mRDT and its routine usage has remained a source of concern to health-care providers in clinical settings [15,16]. Some of the challenges include erratic supply, non-availability of test kits and health care worker's perspectives about its accuracy and reliability with resultant non-adherence to test result when used [15,16]. This may not be totally unconnected with varying reports of sensitivity of different types of mRDT kits ranging from 94.3% in Ibadan [17], 47.0% in Port Harcourt [18], 40.3% in Zamfara [19] to as low as 8.3% in Maiduguri [20]. The Sample size estimation: The number of participants for the study was calculated using the following formula: [21] where: n = minimum number of participants needed; Zα = standard normal deviate corresponding to 2-sided level of significance at 5% = 1.96; p = prevalence of malaria parasitaemia among under-five children presenting with malarial at a general hospital in Ota, Ogun State, 70% [22]. q = 1 -p; d = level of precision at 5%

Recruitment of participants and data collection: A total of
370 febrile under-five children who presented at the emergency and general out-patient (GOP) units of the OMCH with history of fever (axillary temperature > 37.5°C) and a provisional diagnosis of malaria and whose parent/caregivers gave informed consent for the study were recruited consecutively over a period of two months. We excluded all children who were severely ill and because we could not analyze the drug level of those who have taken any anti-malaria drug, they were also excluded. Data on demographic characteristics, child characteristics and episodes of malaria illness were collected from the caregivers using pre-tested interviewer administered questionnaire.
Laboratory examination: Capillary blood sample of each febrile child obtained by finger prick was collected on frosted end slides and CareStart TM Histidine-rich protein 2 (HRP-2) RDT Cassette. Thick and thin blood films were prepared, stained with Giemsa same day and examined microscopically according to WHO standards [23].
Parasite density per microliter of blood (parasitaemia) was determined according to the number of parasites per 200 white blood cells (WBC), assuming a total WBC count of 8,000 /μL and expressed as follows: Parasite density µL -1 = parasite count/number of white blood cells counted × 8000 Microscopic examination of all stained slides was conducted by a trained WHO certified malaria microscopist with 7 years of experience, following standard methods [23]. Each sample was tested with CareStart TM malaria Pf RDT, Lot   [30], Jos by Sheyin and Bigwan [31], Lagos by Ben-Edet et al., [32], Enugu by Oguonu and Okafor [33], and Zamfara by Abdulkadri et al [19], respectively.
The disparity in reported sensitivity may not be because the studies used different kits, for instance while Ajumobi, Falade, and Jeremiah all used SD Bioline malaria Pf RDT, they still reported discordant sensitivity of 100%, 94.3% and 47% respectively. Also for the CareStart TM RDT HRP2 Pf, while the current study gave a sensitivity of 94.6%, Sheyin and Bigwan, and Abdulkadri reported 78.4% and 40.3% respectively. These observed conflicting reports may be due to the functionality of RDTs which remains unable to detect parasites at low densities (< 200-400/ µL). However, other factors such as storage which were not considered in the current study may be responsible for the differences which has eroded the confidence of end users. Although the RDT specificity was high (91.4%), 27 children still had false positive results while microscopy was negative. This may imply that the children did not have active malaria, or had incomplete treatment with antimalarial drug, however, the positive RDT result could be attributed to delayed clearance of antigen from the circulation. It has been shown that malaria antigen clearance could be delayed for up to 4 weeks even, when treatment was successful Abdulkadri et al [19]. High